Armored RNA Quant ™ Development of Armored RNA ® as a Primary Standard

1. Armored RNA Quant™Development of Armored RNA® as a Primary Standard Cindy WalkerPeach, Ph.D. Director of Research and cGMP Operations Ambion Diagnostics, Austin, Texas USA BSI EN ISO9001:2000 Certified Quality System Regulations (21CFR§820) BSI Registration Number: FM 57081 Medical Device Manufacturing Owner/Operator Number: 9049048 SoGAT 2003 June 5, 2003

2. RNA Controls and Standards • Stable – ribonuclease resistant • Homogenous – single, sequence-defined transcript • Quantifiable – known copy number • Compatible – performs broadly with RNA-base viral assay technologies • Flexible – allows for addition to human matrix of choice • Non-infectious – ease in handling and shipping

3. In vivo Armored RNA Packaging System

4. RNA Controls and Standards • Stable – ribonuclease resistant • Homogenous – single, sequence-defined transcript • Directly Quantifiable – known copy number • Compatible – performs broadly with RNA-base viral assay technologies • Flexible – allows for addition to human matrix of choice • Non-infectious – ease in handling and shipping

5. In vitro Armored RNA Packaging System

6. In vitro Armored RNA QuantificationPhosphate Assay • Set up standard curve (concentration vs. OD600), using a phosphate standard from NIST • Digest Armored RNA by phosphodiesterase and alkaline phosphatase to nucleosides and inorganic phosphate • Measure the phosphate concentration of the digested Armored RNA and calculate the value using the standard curve

7. In vitro Armored RNA QuantificationPhosphate Assay Inorganic phosphate standard curve used to determine copy number (4.6 copies) of HIV RNA in each Armored RNA particle

8. In vitro Armored RNA QuantificationHPLC Assay • Set up standard curve (concentration vs. peak area) for standard ribonucleosides (A, U, C, G) by HPLC • Digest Armored RNA, using phosphodiesterase and alkaline phosphate to nucleosides and inorganic phosphate • Measure the nucleoside peak areas by HPLC • Based on the Armored RNA sequence, calculate the concentration of the RNA in the Armored RNA preparation

9. In vitro Armored RNA QuantificationHPLC Assay Determination of RNA concentration by HPLC

10. In vitro Armored RNA QuantificationHPLC Assay HPLC assay standard curves used to determine copy number (4.1 copies) of HIV RNA in each Armored RNA particle

11. In vitro Armored RNA QuantificationComparison Determination of RNA copy number per Armored RNA particle Roche Amplicor Monitor Assay results consistent with those of NIST-traceable phosphate assay and standardized HPLC assay in the quantification of in vitro Armored RNA, within a 20% error range

12. Summary An in vitro Armored RNA® packaging system has been developed to produce Armored RNA that is comprised entirely of a single, defined transcript (target sequence), allowing for direct quantification by two independent, direct, physical methods (NIST-traceable phosphate assay and standardized HPLC assay) Acknowledgements Jun Wang Ginny Lai Marty Badgett Britt Pasloske The project described was supported by grant number AI40529 from the NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH awarding component. Armored RNA® technology was jointly developed by Ambion, Inc. and Cenetron Diagnostics, LLC. Armored RNA® is a registered trademark of Ambion and Cenetron.